Growth may not be required. Over the last decade, culture-independent or molecular methods for identification of foodborne disease (FBD) pathogens in food-safety testing have become more popular. They are faster and accurate, and they let producers more quickly release product or take corrective action. Extending shelf life and safety are often key features behind their uptake.
Although they won’t yet replace culture for growing and isolating pathogens to identify strains, molecular testing technologies exist, and many food testing labs are switching over. Choosing which approach to adopt is easier thanks to an informative white paper from Jani Holopainen, Senior R&D Manager in Molecular Food Safety Microbiology for Thermo Scientific.
What are molecular methods?
Molecular methods use genomic material, usually DNA, to detect FBD pathogens in food matrices. The most common methods in use are thermocycling using quantitative real-time PCR (polymerase chain reaction), or by isothermal amplification. The starting materials for each of these is the DNA of microbial pathogens, which are isolated and then amplified during sample preparation. The white paper gives a good description of each of the methods, including notes on the different methodologies available for each technology.
Real-time PCR uses temperature cycling and polymerase to amplify DNA in the sample while using fluorescent dyes to measure abundance in real time. Dyes can be non-specific, binding to all double-stranded DNA, or probe-based, which measures only target DNA.
SYBR Green fluorescent dye binds non-specifically to all double-stranded DNA. It’s an inexpensive approach, but requires an extra melting step to increase specificity. Since melting temperature relates to DNA sequence and also reduces fluorescence, the curve generated after the PCR cycles helps identify whether the amplified DNA is from the target. This increases the time needed for analysis by 30 to 45 minutes.
Probe-based DNA detection relies on specifically targeting the DNA of interest by creating fluorophore-tagged probes that show when target DNA is amplified. Since multiplexed reactions are possible, internal amplification controls (IACs) can be used to show that the reaction is proceeding properly.
TaqMan™ probes are hydrolysis probes that carry a fluorophore and a quencher. When free, the fluorescent signal is suppressed, activating only when the probe binds to target DNA. Attaching different fluorophores to each specific probe makes it possible to amplify different targets in the same reaction by multiplex PCR.
More complex probe design in molecular beacon probes, Solaris™ and Scorpion™ probes take the fluorophore/quencher assembly a stage further to increase binding specificity, reduce degradation and promote reaction efficiency.
Dual-hybridization or FRET probe pairs carry different fluorescent dyes and bind near each other on the target DNA. The fluorescence resonance energy transfer (FRET) creates the signal when the two probes are close together. Design is more complex, and this technology is also more difficult to multiplex.
Isothermal amplification instruments are less complicated and cheaper than PCR thermocyclers, since reactions do not require change in temperature. However, the complex biochemistry required to successfully amplify target DNA means that multiplexing is complicated. For this reason, using IACs is generally not possible, so it’s difficult to verify that a reaction was successful. Furthermore, approaches such as L-AMP (loop-mediated isothermal amplification) can amplify more non-target DNA through cross-reaction.
Along with molecular approaches and costs, lab managers should also consider instrumentation. Versatility makes for a sensible investment, with open systems providing an instrument that can be used for a variety of tests without restricting users to a single supplier. It’s also worth thinking about connectivity, where easy data uploads speed up results and reduce operator error.
Read more articles and papers discussing food pathogen testing and molecular techniques in our food microbiology learning center
Culture-independent molecular testing: